Aminoglycoside antibiotics are still used despite their well-known ototoxic side-effects. Preliminary data in our lab suggests that different aminoglycosides may act via partially overlapping cell death pathways but that different antibiotics may also activate unique pathways. This research will examine molecular pathways that are differentially involved in neomycin vs. gentamicin-induced hair cell death in the zebrafish lateral line. Specifically, this proposal focuses on the Bcl-2 family of proteins, which contains both pro- and anti- apoptotic members. This proposal tests the hypothesis that Bcl-2 and Bax are specifically involved in neomycin toxicity but not gentamicin toxicity in the zebrafish lateral line. The specific aims of this study are 1) to characterize the distribution of Bcl-2 related proteins in normal zebrafish hair cells and to compare this distribution to hair cells treated with neomycin or gentamicin. 2) To genetically and pharmacologically manipulate Bcl-2 family members in order to study their precise contributions to hair cell death. Aim 1 will be accomplished using immunocytochemistry to localize proteins in different treatment groups. For aim 2, antisense oligonucleotides (morpholinos) will be used for underexpression experiments and transgenics will be created for overexpression studies. Finally, this research will 3) screen two libraries of approved drugs and small drug-like molecules for compounds that specifically modulate gentamicin-induced hair cell death. This gentamicin-specific screen will complement our previous screen using neomycin and should identify new molecular targets that differentially regulate aminoglycoside ototoxicity. These targets represent putative points in apoptotic pathways for therapeutic intervention of aminoglycoside-induced hearing loss and targets of future studies using other ototoxic stimuli such as noise. Lay description: The proposed research uses the zebrafish lateral line system as a whole-animal model to study death of sensory hair cells, the cells responsible for hearing and balance in all vertebrates. This research looks at specific molecules underlying hair cell death in response to antibiotics and will examine ways to prevent this death, potentially providing new therapeutic targets for prevention of hearing loss.